**4. Targeting diabetic complications via targeting epigenetic marks**

Heritable epigenetic alterations are the results of interactions between environmental (momentary) and genetic (long-standing) components and thus, may play a decisive role in the pathophysiology of diabetic complications. They are able to alter the gene expression, thereby, gene function, the underline mechanism in the pathogenesis of vascular complications of diabetes. Reversible attribute of epigenetic marks provides immense opportunity of developing restorative interventions for treating patients with these complications. Till date, some of the drugs targeting epigenetic marks are already being clinically used for cancer therapy including HDAC inhibitors [130] and DNA methylation inhibitors [131, 132]. However, preclinical studies targeting histone as well as DNA methylation are still in progress [133–135]. Metformin, the current line of drug for treating hyperglycemia, upregulates sirtuin 1 (SIRT1) expression along with downregulating NF-κB expression [136], SIRT1 has been shown to possess NAD+ -dependent protein deacetylase activity [137]. In glomerular mesangial cells, SIRT 1 induces antioxidant genes and simultaneously downregulates TGF-β1 and the expression of AGEs-induced fibronectin [138]. In diabetic mice glomeruli, BF175, a SIRT1 agonist, ameliorates hyperglycemia-induced podocyte loss, proving the protective role of SIRT1 against diabetes-induced kidney damage [139]. Recently, angiotensin II (Ang II) of RAAS has been reported to induce the expressions of few non-coding RNAs including miRNAs [140] and lncRNAs [141] as well. Enhancers, the elements that affect transcription of genes and are associated with specific histone modifications [142], when blocked by JQ1, a Bromodomain (an epigenetic reader) inhibitor, also obstructs enhancer functions along with attenuation of Ang II-mediated hypertension and inflammation *in vivo* in vascular smooth muscle cells (VSMCs) [143], hence, strongly supporting the importance of targeting enhancers in Ang II-mediated actions for treating vascular complications. This, in turn, could reveal evidence directing new therapeutic interventions for treatment of diabetic vascular complications. In addition, the modified inhibitor of miR-192 i.e., Locked nucleic acid (LNA) not only downregulates key fibrotic markers of kidney damage but also shown to reduce proteinuria in diabetic mice [144], favoring miRNAs based therapeutic interventions for DN. Several studies have also reported the amelioration of kidney-injury parameters via targeting miR-21 [145–147] implying that its inhibition could be a promising therapeutic intervention in DN. Recently with the use of latest and advanced approach of genome editing i.e., CRISPR-Cas9, locus-specific changes in epigenetic alterations could be generated owing to the fusion of Cas9 proteins with various DNMTs or TETs or histone modification proteins [148–150],

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

Several miRNAs including miR-29, miR-192, miR-194, miR-200b/c, miR-204,

miR-215, miR-216a, miR-217, miR-377 etc. have been found to be associated with DN. Characteristics of DN includes fibrosis, accumulation of extracellular matrix (ECM), podocyte dysfunction and proteinuria [113, 114]. TGF-β has been implicated in the pathogenesis of DN and is found to be upregulated during the progression of DN, which in turn, induce fibrotic events, kidney deterioration and dysfunction [114]. TGF-β has shown to upregulate several miRNAs including miR-192, miR-216a, miR-217 in mesangial cells as well as in kidneys of diabetic mouse models as compared to control group [115–117]. ZEB2, a translation repressor that supress fibrotic gene collagen type 1 Alpha 2 (*Col1a2*), was observed to get suppressed by miR-192, thus, resulted in an increased expression of *Col1a2* gene and contribute to matrix accumulation and kidney fibrosis in DN model [115]. In diabetic mice, increased expressions of p53, TGF-β and miR-192 was reported in renal cortex and was found to be associated with augmented fibrosis and glomerular expansion as compared to control. Moreover, knockout of miR-192 gene resulted in decreased markers of DN. However, conflicting reports to these results are also described. One of such reports observed that TGF-β decreased the expression of miR-192 in cultured proximal tubule cells and concluded that decreased miR-192 levels are associated with increased fibrogenesis in PTCs [118]. Another study also showed that kidney fibrosis was associated with the loss of miR-192 [119]. These contradictory studies showed that the interconnection between DN and miR-192 is much more complicated than it seems. Also, a decreased expression of miR-21 was found in DN and albuminuria was decreased in diabetic mice due to ectopic expression of miR-21 [120]. miR-377 expression was found to be upregulated in DN [121]. It actually alters the levels of MnSOD and PAK1, which in turn, resulted in augmented fibronectin expression in mesangial cells in streptozotocin (STZ) induced diabetic model, thus contributing to DN progression indirectly. TGF-β induced miR-216a expression has been shown to increased collagen (*Col1a2*) expression [116] and subsequently participates in the fibrogenesis in proximal tubular cells (PTCs) [122]. Another important contributor to DN is VEGF and treatment with anti-VEGF showed to improve kidney functions in diabetic animal model [123]. Earlier miRNA-93 was considered as 'signature miRNA' in both *in vivo* as well as *in vitro* hyperglycemic environment [124]. Long et al. also demonstrated that increased expression of miR-93 resulted in reduced high glucose-stimulated

VEGF-A levels via downregulation of the host *MCM7* gene promoter.

Earlier studies have also reported the role of miRNAs in diabetic retinopathy. Neovascularization is the hallmark of DR and several studies have confirmed the importance of miRNAs in neovascularization regulation in retina [125]. Microarray studies recognized increased (miR-146, miR-106a, miR-181, miR-199a, miR-214, miR-424 and miR-451) as well as decreased expressions of various miRNAs (miR-31, miR-150, miR-184) in model of ischemic retinopathy [126]. In retina and retinal endothelial cells (RECs), increased miRNAs corresponding to NF-κB, p53 and VEGF were identified reflecting pathological changes of early DR by means of functional analysis, thus, revealing the role of miRNA in pathogenesis of DR [127]. In diabetes, downregulated miR-200b was detected in retina of diabetic rat model with simultaneous elevated levels of VEGF mRNA and protein. In addition, *in vitro* miR-200b antagonist transfection resulted in elevated VEGF expression [128]. This demonstrates VEGF to be the direct target of miR-200b. During early stage of diabetes, miR-29 shown to be anti-apoptotic for retinal ganglion cells (RGCs) and inner nuclear layer (INL) cells through pro-apoptotic RNA dependent (PKR)

Therefore, this chapter has enlightened the role and contribution of epigenetic mechanisms in the pathogenesis of two major diabetic vascular complications i.e.,

**184**

signaling pathway [129].

thus, reversing the epigenetic marks of important genes involved in the pathogenesis of s disease. Despite extensive ongoing research, more detailed epigeneticstargeted approach is required to combat diabetic microvascular complications.
